The Autophagy Protein ATG9A Promotes HIV-1 Infectivity Nef is an accessory protein encoded by the primate immunodeficiency viruses HIV-1, HIV-2 and SIV that conttirbutes to viral replication, assembly, budding, infectivity and immune evasion, through engagement of various host cell pathways. To gain a better understanding of the role of host cell proteins in the functions of Nef, we carried out tandem affinity purification-mass spectrometry analysis, and identified over 70 HIV-1 Nef-interacting proteins, including the autophagy-related 9A (ATG9A) protein. ATG9A is a transmembrane component of the machinery for autophagy, a catabolic process in which cytoplasmic components are degraded in lysosomal compartments. Pulldown experiments demonstrated that ATG9A interacts with Nef from not only HIV-1 and but also SIV. However, expression of HIV-1 Nef had no effect on the levels and localization of ATG9A, and on autophagy, in the host cells. To investigate a possible role for ATG9A in virus replication, we used CRISPR/Cas9 to knock out (KO) ATG9A in HeLa cervical carcinoma and Jurkat T cells, and analyzed virus release and infectivity. We observed that ATG9A KO had no effect on the release of wild-type (WT) or Nef-defective HIV-1 in these cells. However, the infectivity of WT virus produced from ATG9A-KO HeLa and Jurkat cells was reduced by 4-fold and 8-fold, respectively, relative to virus produced from WT cells. This reduction in infectivity was independent of the interaction of Nef with ATG9A, and was not due to reduced incorporation of the viral envelope (Env) glycoprotein into the virus. The loss of HIV-1 infectivity was rescued by pseudotyping HIV-1 virions with the vesicular stomatitis virus G glycoprotein. From these studies, we concluded that ATG9A promotes HIV-1 infectivity in an Env-dependent but Nef-independent manner. ATG9A could promote infectivity by participating in either the removal of a factor that inhibits infectivity or the incorporation of a factor that enhances infectivity of the viral particles. ATG9A is thus a novel host cell factor implicated in HIV-1 infectivity.